Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Messenger RNA Destabilization by -1 Programmed Ribosomal Frameshifting(2012) Belew, Ashton Trey; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although first discovered in viruses, previous studies have identified programmed -1 ribosomal frameshifting (-1 PRF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. This work improves and extends the computational methods used to search for potential -1 PRF signals. It continues to examine four yeast -1 PRF signals and show that they promote significant mRNA destabilization through the nonsense mediated (NMD) and no-go (NGD) decay pathways. Yeast EST2 mRNA is highly unstable and contains up to five -1 PRF signals. Ablation of the -1 PRF signals or of NMD stabilizes this mRNA. These same computational methods identified an operational programmed -1 ribosomal frameshift (-1 PRF) signal in the human mRNA encoding CCR5. A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay (NMD) pathway. CCR5-mediated -1 PRF is stimulated by at least two miRNAs, one of which is shown to directly interact with the CCR5 -1 PRF signal. Structural analyses reveal a complex and dynamic mRNA structure in the -1 PRF signal, suggesting structural plasticity as the underlying biophysical basis for regulation of -1 PRF.Item Characterization of Arabidopsis thaliana SR protein genes: mutations, alternative splicing, and ESE selection(2007-06-07) edmonds, jason matthew; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA processing in eukaryotes is a highly complex process requiring numerous steps and factors that can play roles in the regulation of functional protein production. SR proteins are a well-defined family of splicing factors identified by a conserved RNA Recognition Motif (RRM) and carboxyl-terminal arginine/serine (RS) repeats. SR proteins are known to bind to mRNA precursors via Exonic Splicing Enhancers, and to recruit U2AF and the U1 snRNP to promote splicing. I have identified mutations in five Arabidopsis thaliana SR protein genes that result in altered phenotypes. Two (scl28-1 and srp31-1) result in embryonic lethal phenotypes, while three others (sc35-1, sr45-1, and srp30-1) result in viable and fertile plants with a range of phenotypes. I have also found that mutations in individual SR protein genes can effect the ability of a specific sequence to act as an ESE and hence affect splicing efficiency. Because 16 of the 20 Arabidopsis thaliana SR proteins themselves are alternatively spliced, I have looked for cross regulation using RT-PCR analysis of isoform accumulation in alternatively spliced SR protein genes. I found that SR proteins do, in fact, regulate the alternative splicing of gene targets and do so in both a gene and a tissue specific manner. In order to begin to fully understand the relationship between individual SR proteins it is essential to know when and where they are expressed throughout development. I have studied the expression pattern of 16 of the 20 SR proteins in the roots of wild-type plants as well as sc35-1, srp30-1, and sr45-1 mutants. I have identified both spatial and temporal expression patterns for these 16 proteins relative to specific tissues that compose the root.